Purpose:

Androgen deprivation regenerates the thymus in adults, expanding of T-cell receptor V β repertoire in blood and lymphoid organs and tumor-infiltrating lymphocytes in human prostate tumors. In melanoma murine models, androgen receptor promotes metastases and androgen blockade potentiates antitumor vaccine efficacy. This phase I study evaluated the safety, efficacy, and pharmocodynamics of androgen deprivation with the gonadotropin releasing hormone (GnRH) agonist triptorelin combined with nivolumab in male patients with melanoma resistant to anti–PD-1.

Patients and Methods:

Adult male patients with advanced melanoma who progressed under anti–PD-1 containing regimens received triptorelin 3.75 mg every 4 weeks, nivolumab 3 mg/kg every 2 weeks, and bicalutamide 50 mg once daily during the first 28 days. Tumor response was first assessed after 3 months; adverse events (AE) were monitored throughout the study. T-cell receptor excision circles (TREC), a biomarker of thymus activity, were explored throughout the study.

Results:

Of 14 patients, 4 were locally advanced and 10 had distant metastases. There were no grade 4 or 5 AEs. Five grade three AEs were reported in 4 patients. According to RECIST v1.1, best overall response was partial response (PR) in one patient with a pancreas metastasis, stable disease (SD) in 5 patients, and progressive disease in 8 patients. According to iRECIST, a second PR occurred after an initial pseudoprogression, TRECs increased in 2 patients, one with PR who also had an increase in TILs, and the second with SD.

Conclusions:

This combination was well tolerated. Disease control was obtained in 42.8% (RECIST) and 50% (iRECIST). The evidence for thymus rejuvenation was limited.

Translational Relevance

Almost half of the patients become resistant and there is a high medical need to identify an effective treatment for patients who have progressed under PD1-based regimen. Most patients who do not respond to immunotherapy have tumors devoid of tumor-infiltrating lymphocytes (TIL). Melanoma cells harbor androgen receptors. Both in human and animal models, androgen deprivation regenerates the thymus in adults. Combining androgen deprivation to nivolumab in 14 male patients with melanoma presenting resistance to anti–PD-1 provided disease control in 42.8% (RECIST) and 50% (iRECIST) and thymus rejuvenation in 2 patients, including 1 with PR, together with augmentation of TILS. These findings suggest that blocking AR in combination with ICI is a promising therapeutic strategy that should be further explored in both male and female patients.

In spite of the major advances in the field of melanoma treatment using checkpoint inhibitors, almost half of the patients become resistant and there is a high medical need to identify an effective treatment for patients who have progressed under PD1-based regimen (1). Most patients who do not respond to immunotherapy have tumors devoid of tumor-infiltrating lymphocytes (TIL), called “cold tumors,” as a consequence of immunotolerance or “immune-ignorance” (2). One of the goals of treatments aiming at reversing resistance to PD1 blockade is thus to boost anticancer immune response by increasing the number of TILs.

For many years, several epidemiologic studies suggested that gender is an independent determinant of melanoma outcome with females having a significantly better prognosis than males among patients with melanoma across melanoma stages and treatment types (3–7). The presence of androgen receptors (AR) in human melanoma samples is associated with a poor prognosis compared with AR-negative samples (7, 8). Blockade of AR in experimental melanoma improved response to BRAF/MEK inhibitors (7). In a murine melanoma model, it was shown that AR can promote melanoma metastasis via altering the miRNA-539–3p signal (8).

Besides the stimulation of melanoma through AR receptors, an immunosuppressive effect of androgens was further demonstrated by showing that activation of T-cell androgen receptors with testosterone leads to the upregulation of the protein phosphatase PTPN1 that blocks T-cell differentiation (9). In addition, testosterone blocks the differentiation of T helper (Th) 1 and Th17 cells (9) while stimulating Th2 cells (10, 11), upregulating the expression of Foxp3 (12), and expanding T regulatory cells (13).

Androgen deprivation can be obtained by desensitisation of pituitary gonadotropin-releasing hormone (GnRH) receptors with continuous administration of GnRH agonist (GnRH-A) that, by suppressing the luteinizing hormone and follicle stimulating hormone, reduces testosterone to castrate levels (14).

Interestingly, androgen suppression by GnRH-A was shown to quickly restore the thymus and its function in adult male rats and mice (15, 16). In male mice, treatment with GnRH-A increased the number of CD4+ and CD8+ T-cells in blood and lymph nodes (17). In patients with prostate cancer, GnRH-A treatment resulted in thymus rejuvenation, as shown by the increase in the molecular recombination marker T-cell receptor excision circles (TREC), a reliable marker of newly formed T cells in the circulation (16). In prostate cancer, AR blockade enhanced CD8+ T cells activity and produced improved response to ICI (18). In addition, GnRH-A treatment decreased the immunosuppressive phosphatase PTPN1 (9) prevented radiotherapy-induced lymphopenia (19), increased circulating CD4+ and CD8+ T cells with expansion of naïve and memory T cells as well as natural killer cells (19). GnRH-A treatment also increased TILs in prostate cancer samples with induction of an oligoclonal response (20). After allogenic or autologous stem cell transplantation, temporary GnRH-A treatment resulted in enhanced immune system regeneration in both males and females as illustrated by a long-term increase in TRECs levels in both sexes (21).

These effects might be in part due to a direct action of GnRH-A on immune cells. Indeed, by binding to GnRH receptors on the surface of human, porcine, and rat lymphocytes, GnRH-A increases lymphocyte proliferation upon mitogen or cytokine stimulation, and upregulates the expression of IL2 receptors in human and mouse spleen lymphocytes and thymocytes (22–25). In addition, administration of GnRH-A to orchiectomized rats could efficiently restore age-linked decrease of thymus weight, as well as thymocyte proliferative capacity with a two-fold increase in thymocyte proliferation compared with orchidectomy alone (16), suggesting a direct effect on T cells.

Altogether, the considerations above are strong incentive to evaluate the addition of androgen blockade using a GnRH-A to immune checkpoint blockade in patients with melanoma. We hypothesized that the GnRH-A triptorelin could overcome resistance to anti-PD1 inhibitors by inducing thymus regeneration and production of naïve T cells with large T-cell receptor repertoire that could potentially recognize tumor antigens. We further hypothesized that the addition of triptorelin to nivolumab would not significantly increase immune-related toxicity.

Here we report the results of a phase I study aimed at evaluating the safety of triptorelin in combination with nivolumab and bicalutamide, and the potential of this combination to reverse resistance to PD-1 inhibitors in male patients with melanoma pretreated with anti-PD1.

Study design

Debio 8200-IMM-101 was a phase I, single-arm, open-label study performed at six centers in France. The study protocol and all amendments were approved by the French ethics committee (Comité pour la Protection des Personnes) and the National Agency for the Safety of Medicines and Health Products. The protocol was conducted in accordance with the Declaration of Helsinki and Good Clinical Practice Guidelines. All patients provided written informed consent before screening.

The primary objective of the study was to assess the safety and tolerability of a fixed dose of triptorelin when given in combination with the approved standard dose of nivolumab and bicalutamide in male patients with refractory/relapsing advanced/metastatic melanoma. Secondary objectives included the evaluation of anti-tumor activity and pharmacodynamic effects of triptorelin when combined with nivolumab and bicalutamide, and the assessment of the pharmacokinetics of triptorelin when combined with nivolumab and bicalutamide.

Patients

The study population consisted of male patients ages ≥18 years with refractory/relapsing locally advanced or metastatic histologically confirmed melanoma, progressing under anti–PD-1/PD-L1 containing regimens (with or without anti-CTLA-4). Patients had to agree to collection of paired tumor biopsies, including one at screening. Other key inclusion criteria included measurable lesions according to RECIST v1.1 (26), ≤2 previous therapy lines with an anti–PD-1/PD-L1 containing regimen; Eastern Cooperative Oncology Group performance status of 0 to 1; serum lactate dehydrogenase <2× upper limit of normal; adequate haematological, renal, hepatic, and pulmonary functions; serum testosterone concentrations within the normal reference for the age of the patient. Notably, patients with completed primary treatment of brain metastases were to be clinically stable, asymptomatic and off steroids for at least 28 days.

Key exclusion criteria included history of immune-related toxicity from a previous anti–PD-1 containing regimen leading to permanent treatment interruption; history of organ transplant; history of primary immunodeficiency; previous systemic corticosteroid therapy >10 mg/day within 14 days prior to first drug administration; history of autoimmune disease (with a few exceptions); vaccination within the 4 weeks prior to the first study drug administration; evidence of active, noninfectious pneumonitis or history of interstitial lung disease.

Treatment

Treatment consisted of triptorelin embonate 3.75 mg sustained-release formulation intramuscularly every 4 weeks and nivolumab (Opdivo) 3 mg/kg intravenously every 2 weeks. Bicalutamide (Casodex) 50 mg was taken orally daily for the first 28 days to counteract the initial testosterone peak (“flare”) expected after the first triptorelin administration. Dose reductions were not permitted for any study drugs; interruption of nivolumab treatment was permitted for a maximum of 2 weeks in case of toxicity. The planned treatment duration was 48 weeks (i.e., 12 triptorelin cycles of 4 weeks). In patients deriving benefit, treatment could continue for up to 12 cycles until disease progression, unacceptable toxicity, withdrawal of consent, or premature termination of the study, whichever came first. Patients with disease progression after three cycles (at Week 11 or 12) had to be reassessed 7 to 8 weeks later (at Week 19 or 20). If progression was confirmed, study treatment was to be stopped, unless it was considered beneficial to the patient to continue the therapy up to 12 cycles.

Treatment extension was offered to patients who had completed Cycle 12 and might have benefited from a continuation of the combination treatment.

Study assessments and statistical analysis

Adverse events (AE) and serious AEs (SAE) were monitored throughout the study until 30 days after end of treatment (EOT). AEs were coded according to the Medical Dictionary for Regulatory Activities version 21.1 and graded according to the National Cancer Institute Common Terminology Criteria for Adverse Events version 4.03.

Safety haematology measurements were performed at screening and every 2 weeks thereafter predose from Day 1 until EOT and were analyzed locally.

Evaluation of tumor response was performed after three triptorelin cycles, at Week 11 or 12, according to RECIST v1.1 (26).

Blood samples for triptorelin pharmacokinetic assessments were collected on Days 1 and 57, predose and at 1 hour (i.e., just before nivolumab infusion), 2 hours, and 4 hours postdose; at predose of any study drugs on Days 15, 29, 71, 85, 169, 253; and at EOT (Day 337). Serum triptorelin levels were measured by LC/MS-MS.

Blood samples were collected on Days 1, 15, 29, 57, 71, 85, 169, 253, and at EOT (Day 337) to evaluate the effect of treatment on testosterone and TRECs. Serum testosterone levels were measured by LC/MS-MS. TRECs in peripheral blood mononuclear cells (PBMC) were measured by qPCR using the MyTREC Sensi Duplex TREC/Beta Actin Real-Time qPCR Assay Kit (GenenPlus, GP-D3012096).

Tumor biopsies were collected at baseline during screening and at cycle 3 (Week 11 or 12) from the same lesion or nearby lesions of the same tissue type prior to CT scan/MRI. Density of TILs (CD3+, CD8+, CD163+, FoxP3+, CD68+, and PD-L1+ cells) was assessed by IHC. Only patients with assessments performed on biopsies collected from the same lesion or nearby lesions of the same tissue type at both baseline and at Cycle 3 were assessed for changes from baseline.

All statistical analyses were descriptive due to the small number of patients. Statistical analysis populations comprised the safety population (patients who received at least one dose of any study drug), which was also used for the analysis of pharmacodynamic data, efficacy population (patients with tumor assessment according to RECIST v1.1 at baseline and at least once during treatment) and pharmacokinetics population (patients who received at least one dose of triptorelin and who had at least one triptorelin serum concentration value).

Data availability

The data generated in this study are available on request to the corresponding author.

Patient characteristics

Between January 5 and October 30, 2018, 14 male patients with a mean (range) age of 65 (45–82) years were enrolled and treated (Table 1; Supplementary Tables S1 and S2). All patients had been treated with anti–PD-1 as last therapy before entering the study. Eight patients were primary refractory to anti–PD-1 therapy and 6 were secondary refractory after initial response to anti–PD-1 therapy. One patient had been previously treated with anti BRAF/MEK therapy. Twelve patients had cutaneous melanomas (2 of which with unknown primary location) and 2 patients had sinonasal mucosal melanomas. Among the patients with cutaneous primaries, 4 had advanced stage III, whereas 9 had stage IV distant metastases. One mucosal patient with melanoma had a locally advanced nonresectable disease and one also had visceral metastases (stage IV).

Table 1.

Patient characteristics, safety population.

Number of patients 14 
Age, year 
 Mean (range) 64.9 (45–82) 
Race 
 White, n (%) 11 (78.6) 
 Black, n (%) 3 (21.4) 
Cutaneous melanoma, n (%) 12 (85.7) 
Mucosal melanoma, n (%) 2 (14.3) 
Stage III, n 4 
 M0, N2c, n 
 M0, N3c, n 
Stage IV, n 10 
 M1c, N0, n 
 M1c, N2b, n 
 M1c, N2c, n 
 M1a, N3c, n 
 M1c, N3c, n 
 M1d, N3c, n 
 M1b, N3c, n 
Number of patients 14 
Age, year 
 Mean (range) 64.9 (45–82) 
Race 
 White, n (%) 11 (78.6) 
 Black, n (%) 3 (21.4) 
Cutaneous melanoma, n (%) 12 (85.7) 
Mucosal melanoma, n (%) 2 (14.3) 
Stage III, n 4 
 M0, N2c, n 
 M0, N3c, n 
Stage IV, n 10 
 M1c, N0, n 
 M1c, N2b, n 
 M1c, N2c, n 
 M1a, N3c, n 
 M1c, N3c, n 
 M1d, N3c, n 
 M1b, N3c, n 

Median (range) duration of previous immunotherapy was 6 (1–38) months.

Safety

The majority of patients (78.6%) completed at least three treatment cycles, and 3 (21.4%) patients completed 12 cycles.

The most commonly reported TEAEs (Table 2; Supplementary Table S3) were hot flushes (in 6 [42.9%] patients), asthenia, and constipation (each in 4 [28.6%] patients). The severity and frequency of these AEs were in line with those found in patients with prostate cancer during ADT due to changes in testosterone levels.

Table 2.

TEAEs reported in >1 patient by SOC and PT – safety population.

SOC, n (%)Total
PT, n (%)(N = 14)
Gastrointestinal disorders 9 (64.3) 
 Constipation 4 (28.6) 
 Diarrhea 3 (21.4) 
 Nausea 3 (21.4) 
 Abdominal pain 2 (14.3) 
General disorders and administration site conditions 8 (57.1) 
 Asthenia 4 (28.6) 
Vascular disorders 8 (57.1) 
 Hot flush 6 (42.9) 
 Hypertension 2 (14.3) 
Skin and subcutaneous tissue disorders 7 (50.0) 
 Pruritus 3 (21.4) 
 Dry skin 2 (14.3) 
 Vitiligo 2 (14.3) 
Investigations 5 (35.7) 
 Blood creatine phosphokinase increased 2 (14.3) 
 Weight decreased 2 (14.3) 
Musculoskeletal and connective tissue disorders 5 (35.7) 
 Back pain 2 (14.3) 
Respiratory, thoracic, and mediastinal disorders 5 (35.7) 
 Dyspnea 2 (14.3) 
Blood and lymphatic system disorders 4 (28.6) 
 Anemia 2 (14.3) 
Neoplasms benign, malignant, and unspecified (including cysts and polyps) 4 (28.6) 
 Tumor pain 3 (21.4) 
Psychiatric disorders 4 (28.6) 
 Depression 2 (14.3) 
Metabolism and nutrition disorders 3 (21.4) 
 Decreased appetite 3 (21.4) 
SOC, n (%)Total
PT, n (%)(N = 14)
Gastrointestinal disorders 9 (64.3) 
 Constipation 4 (28.6) 
 Diarrhea 3 (21.4) 
 Nausea 3 (21.4) 
 Abdominal pain 2 (14.3) 
General disorders and administration site conditions 8 (57.1) 
 Asthenia 4 (28.6) 
Vascular disorders 8 (57.1) 
 Hot flush 6 (42.9) 
 Hypertension 2 (14.3) 
Skin and subcutaneous tissue disorders 7 (50.0) 
 Pruritus 3 (21.4) 
 Dry skin 2 (14.3) 
 Vitiligo 2 (14.3) 
Investigations 5 (35.7) 
 Blood creatine phosphokinase increased 2 (14.3) 
 Weight decreased 2 (14.3) 
Musculoskeletal and connective tissue disorders 5 (35.7) 
 Back pain 2 (14.3) 
Respiratory, thoracic, and mediastinal disorders 5 (35.7) 
 Dyspnea 2 (14.3) 
Blood and lymphatic system disorders 4 (28.6) 
 Anemia 2 (14.3) 
Neoplasms benign, malignant, and unspecified (including cysts and polyps) 4 (28.6) 
 Tumor pain 3 (21.4) 
Psychiatric disorders 4 (28.6) 
 Depression 2 (14.3) 
Metabolism and nutrition disorders 3 (21.4) 
 Decreased appetite 3 (21.4) 

Note: AEs are coded according to the version 21.1 of MedDRA. A patient is counted only once at the worst CTCAE grade for multiple events within the same PT/SOC. TEAEs are all AEs starting or worsening during the on-treatment period.

Abbreviations: MedDRA, Medical Dictionary for Regulatory Activities; PT, preferred term; SOC, system organ class; TEAE, treatment emergent adverse event.

However, fatigue and constipation are also commonly reported with nivolumab monotherapy. There were no unexpected drug-related serious or Grade 3 TEAEs. No AEs at the injection site of triptorelin were reported. The combination of triptorelin, bicalutamide (first 4 weeks), and nivolumab did not increase the immune-related toxicity compared with that expected of nivolumab alone.

There were no grade 4 or 5 AEs. Five grade 3 AEs were reported in 4 patients: neutropenia, asthenia, back pain, abdominal pain, and bone pain. Grade 3 neutropenia was attributed to nivolumab and resolved following a treatment interruption of 14 days. The event of abdominal pain was attributed to triptorelin, classified as SAE, and resolved after 39 days without causing treatment interruption (as it started on the day the treatment was discontinued due to disease progression). Of note, both treatment-related events were expected as they are described on the labels of the two products. Five SAEs were reported in 4 patients: the aforementioned event of abdominal pain and an event of back pain (both of grade 3) reported in the same patient, and events of sinusitis, neoplasm progression, and epistaxis.

Efficacy

According to RECIST v.1.1, best overall response (BOR) was assessed as one partial response (PR) in a patient with pancreas metastasis (reduction of 76% from Baseline), five stable diseases (SD) and eight progressive diseases (PD). One patient with extensive sinonasal melanoma was not evaluable for efficacy as protocol therapy was discontinued after 1 month due to major progression. According to a post hoc analysis based on iRECIST criteria (27), a second PR occurred after an initial pseudoprogression in a patient with two inguinal lymph node metastases (reduction of 32% from Baseline; Fig. 1A and B; Supplementary Table S3) as well as a complete disappearance of new cutaneous metastasis. Following withdrawal due to premature termination of the main study by the Sponsor on Study Day 239 (Cycle 9), this patient received 11 additional treatment cycles because the investigator considered that the combination treatment was beneficial. Among the patients with objective response, 1 with PR had a previous PR to pembrolizumab and 1 with SD had a previous PR to pembrolizumab randomized with or without epacadostat (Supplementary Table S2).

Figure 1.

A, Percentage change in tumor size over time, efficacy population (n = 13). B, Best percentage change in tumor size during the study, efficacy population (n = 13). One patient experienced PR after initial progression (pseudoprogression). BOR, best overall response; PD, progressive disease; PR, partial response.

Figure 1.

A, Percentage change in tumor size over time, efficacy population (n = 13). B, Best percentage change in tumor size during the study, efficacy population (n = 13). One patient experienced PR after initial progression (pseudoprogression). BOR, best overall response; PD, progressive disease; PR, partial response.

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TRECs

TRECs levels at baseline and EOT by age category (Fig. 2A <70; Fig. 2,B >70) showed high variability across patients. Increases in TRECs levels from baseline were observed in 2 patients (Fig. 2,B): in a 71-year-old patient with PR as BOR, at all timepoints except EOT, with the highest value (630.3 μg/DNA) at Day 57; in a 72-year-old patient with SD as BOR, at Days 15, 57, 71, 85, and 169, with the highest value (464.5 μg/DNA) at Day 85.

Figure 2.

TREC levels according to age category (A, <70; B, >70) and RECIST response. Changes (%) in TREC levels from baseline by patient and BOR, safety population (n = 14). BOR, best overall response.

Figure 2.

TREC levels according to age category (A, <70; B, >70) and RECIST response. Changes (%) in TREC levels from baseline by patient and BOR, safety population (n = 14). BOR, best overall response.

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TILs

Increases in TILs were observed for 2 patients out of 4 with paired biopsies: one patient with pancreas metastasis and PR as BOR (increase in CD3+, CD8+, Foxp3+, CD163+, and CD68+ cells), and the other with PD as BOR (increase in CD3+ and CD8+ cells, and slight increase in Foxp3+and CD68+ cells; Fig. 3; Supplementary Table S4). The number of TILs was stable in 2 patients with SD.

Figure 3.

Changes in TILs’ density at baseline and after cycle 3, by patient and BOR. CD3+ cells; CD8+ cells; CD163+ cells; FOXP3+ cells; CD68+ cells; subjects with evaluable paired biopsies of safety population (n = 3–4 depending on the biomarker tested). BOR, best overall response.

Figure 3.

Changes in TILs’ density at baseline and after cycle 3, by patient and BOR. CD3+ cells; CD8+ cells; CD163+ cells; FOXP3+ cells; CD68+ cells; subjects with evaluable paired biopsies of safety population (n = 3–4 depending on the biomarker tested). BOR, best overall response.

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Blood lymphocytes

For all patients regardless of their BOR, blood lymphocytes were close to the lower limit of normal range at baseline and during the study with no significant change.

Triptorelin pharmacokinetics

After the first intramuscular injection, triptorelin serum levels increased rapidly, reaching mean geometric maximum serum concentration (Cmax) of 13,892 ng/L with a median (range) time to reach maximum serum concentration (tmax) of 1.1 (1–4) hours. Similar Cmax and tmax values were observed after the third triptorelin injection, indicating no accumulation after repeated dosing. Triptorelin exposure was similar in presence (Days 1–28 in Cycle 1 only) and absence (all subsequent cycles) of bicalutamide treatment. Overall, the pharmacokinetic profile of triptorelin was consistent with data obtained previously in healthy male volunteers with triptorelin 1-month formulation alone (data not shown).

Testosterone

Serum testosterone decreased after treatment initiation (Fig. 4). Mean (SD) testosterone concentrations decreased from 12.2 (5.6) nmol/L at predose Baseline to 0.7 (0.7) nmol/L at EOT.

Figure 4.

Serum testosterone levels at baseline and during study treatment, safety population (n = 14). The plus symbol + denotes the mean at each timepoint; the top and bottom lines of each box denote the 75th and 25th percentiles; the horizontal line between them denotes the median. The whiskers denote 1.5 times the interquartile range, and values outside this range are represented by circles. Nobs, number of observations at timepoint.

Figure 4.

Serum testosterone levels at baseline and during study treatment, safety population (n = 14). The plus symbol + denotes the mean at each timepoint; the top and bottom lines of each box denote the 75th and 25th percentiles; the horizontal line between them denotes the median. The whiskers denote 1.5 times the interquartile range, and values outside this range are represented by circles. Nobs, number of observations at timepoint.

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This is the first study on the combination of androgen deprivation therapy with immune checkpoint inhibition in ICI-resistant melanoma patients. Patients were treated with the monthly dose of triptorelin approved to induce and maintain chemical castration in patients with prostate cancer combined with nivolumab and bicalutamide (only for the first treatment cycle) at the recommended dosage regimens.

The combination of triptorelin, bicalutamide, and nivolumab was well tolerated and did not increase the expected immune-related toxicity of nivolumab. No grade 4 or 5 AEs were reported. Only 2 grade 3 AEs (14%) were considered related to study drugs: abdominal pain and neutropenia, attributed to triptorelin and nivolumab, respectively. Of note, both events were expected as described on the labels of the respective products, and both resolved.

Our study hypothesis was that androgen deprivation would result in thymus rejuvenation and increase in TILs. We monitored TRECs that are known to be released upon rearrangement of the T-cell receptor and to constitute reliable markers of newly formed circulating T cells that are associated with thymus regeneration. we found that 2 of 14 patients had increased levels of TRECs in PBMCs: 1 patient who experienced PR of pancreas metastasis and 1 with SD. Unfortunately, in the patient with PR according to iRECIST, TRECs levels were not assessed during either the pseudoprogression period, or the PR period.

Three months after the first triptorelin injection, TILs were increased in 2 patients. The aforementioned patient with PR of pancreas metastasis with increased TRECs, had increased TILs of different phenotypes (CD4+ cells, CD8+ cells, T regulatory cells) as well as macrophages. Another patient who experienced fast progression and no increase in TRECs, exhibited increase in CD4+ and CD8+ cells. No change in TILs were observed in 2 patients with SD. Because of the small number of patients with data at both Baseline and Cycle 3 (3 or 4 patients, depending on the marker tested), the TILs data are difficult to interpret.

Previous studies in various settings including melanoma have clearly reported immune effects of GnRH-A on TRECs and TILs. In a study of treatment-naïve advanced patients with prostate cancer, GnRH-A treatment resulted in a >25% increase in TRECs levels in PBMCs in 6 of 10 patients (16). In another study, patients with localized prostate cancer treated with GnRH-A at different times before surgery exhibited a progressive increase in TILs with a plateau at Week 3 (20). After the implementation of our study protocol, it was reported that 3 of 10 patients with prostate cancer resistant to the anti-androgen enzalutamide, had responses after adding pembrolizumab (28). These observation are consistent with recent striking results demonstrating a direct immunostimulating action of AR blockade with increase of CD8+ IFNγ secretion and a synergistic effect between AR blockade and PD1 blockade in a murine prostate cancer (ref 18; https://www.nature.com/articles/s41586–022–04522–6).

However, prostate cancer is androgen-dependent and a synergistic effect between PD1 blockade and shedding of tumor-associated antigens due to androgen-deprivation could be involved. But in melanoma cells can also harbor AR and It was even shown that mice of both sexes receiving BRAF/MEK inhibitors had improved response upon AR blockade (7).

We questioned whether the clinical benefit observed in half of our patients could be due to an anti-PD1 rechallenge effect. Indeed, in two small series of 8 patients with melanoma who had been retreated after interruptions of various ICI therapies were found to respond to a rechallenge with anti-PD1 therapy (29, 30). It is important to note that for those patients, reintroduction of a PD-1 inhibitor occurred after a various time interval (range 0.3–17.3 months) during which most patients received other therapies such as chemotherapy or radiotherapy, compared with our study, during which no other anticancer therapies were given (range 30–180 days). Therefore, although we cannot totally rule it out, it is very unlikely that the clinical benefit of the combination triptorelin and nivolumab in our patients could be related to nivolumab only.

In addition, our patients had undergone several prior treatments with various ICIs to which they had become resistant after relatively long treatment durations (median of 6 months; Supplementary Table S2).

One additional reason why we did not observe a higher level of thymus regeneration and better clinical outcome is that our patients had low lymphocyte counts that could not be significantly improved by the combination of triptorelin and nivolumab. Even in the 2 patients with signs of thymus function upregulation, the peripheral lymphocyte counts did not increase.

The representativeness of Study Participants is shown in Supplementary Table S5.

Our study did not include females. However, it was shown that melanoma cells upregulate AR and could undergo better response to targeted therapy after AR blocking (7). This finding would suggest to include females in future trials of AR blockade.

Conclusion

The combination of nivolumab and androgen deprivation with triptorelin is well tolerated in male patients with advanced melanoma. It was associated with a disease control in 6 of 13 evaluable patients (46.1%) but objective responses were seen only in 1 and 2 patients according to RECIST or iRECIST criteria, respectively. Thymus regeneration was found in 2 patients including one responding patient who also had increased TILs, in line with our working hypothesis that triptorelin and/or chemical castration could restore the thymic function and increase TILs.

A randomized controlled study evaluating anti-PD1+ triptorelin versus anti-PD1 alone would provide the opportunity to determine the relative importance of triptorelin and nivolumab and to investigate more response biomarkers.

C. Robert reports personal fees from Sanofi, AstraZeneca, Pierre-Fabre, Roche, Novartis, BMS, MSD, and Ribonexus during the conduct of the study. C. Lebbé reports grants and personal fees from BMS and Roche and personal fees from MSD, Novartis, Amgen, Merck Serono, Sanofi, Pierre-Fabre, Pfizer, Incyte, Avantis Medical Systems, and InflaRx outside the submitted work. T. Lesimple reports personal fees and nonfinancial support from MSD and Pierre Fabre Onc. and personal fees from BMS and Novartis outside the submitted work. V. Nicolas reports personal fees from Debiopharm International SA outside the submitted work. B. Gavillet reports a patent for WO 2019/122998 A1 pending. B. Baroudjian reports personal fees from BMS, Pierre Fabre, Sanofi, and MSD outside the submitted work. F.J. Lejeune reports a patent for WO 2019/122998 A1 pending. No disclosures were reported by the other authors.

C. Robert: Validation, investigation, writing–review and editing. C. Lebbé: Validation, investigation. T. Lesimple: Investigation. E. Lundström: Resources, data curation, formal analysis, validation, writing–review and editing. V. Nicolas: Data curation. B. Gavillet: Data curation. P. Crompton: Data curation, software, formal analysis. B. Baroudjian: Investigation. E. Routier: Data curation, investigation. F.J. Lejeune: Conceptualization, formal analysis, supervision, funding acquisition, validation, methodology, writing–original draft, project administration, writing–review and editing.

Dr. B. Dreno (CHU Nantes, France), Dr. S. Dalle (CHU Lyon, France), and Dr. N. Meyer (Oncopole, Toulouse, France) are acknowledged for their participation in this study. The involvement of Dr. S. Brienza (Paris) and Dr. J.M. Dumont (Debiopharm) in the design of this study is acknowledged. We are grateful to Dr. P. Boyle (Monash University) for having inspired and advised us. We are grateful to the patients who participated in this study and their families. This study was funded by Debiopharm International SA.

Note: Supplementary data for this article are available at Clinical Cancer Research Online (http://clincancerres.aacrjournals.org/).

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